Circular dichroism changes in galactosyltransferase upon substrate binding

118

104

␤-1,4-Galactosyltransferase 1 (Gal-T1) transfers galactose (Gal) from UDP-Gal to N-acetylglucosamine (GlcNAc), which constitutes its normal galactosyltransferase (Gal-T) activity. In the presence of ␣-lactalbumin (LA), it transfers Gal to Glc, which is its lactose synthase (LS) activity. It also transfers glucose (Glc) from UDPGlc to GlcNAc, constituting the glucosyltransferase (Glc-T) activity, albeit at an efficiency of only 0.3-0.4% of Gal-T activity. In the present study, we show that LA increases this activity almost 30-fold. It also enhances the Glc-T activity toward various N-acyl substituted glucosamine acceptors. Steady state kinetic studies of Glc-T reaction show that the K m for the donor and acceptor substrates are high in the absence of LA. In the presence of LA, the K m for the acceptor substrate is reduced 30-fold, whereas for UDP-Glc it is reduced only 5-fold. In order to understand this property, we have determined the crystal structures of the Gal-T1⅐LA complex with UDP-Glc⅐Mn 2؉ and with N-butanoyl-glucosamine (N-butanoyl-GlcN), a preferred sugar acceptor in the Glc-T activity. The crystal structures reveal that although the binding of UDP-Glc is quite similar to UDPGal, there are few significant differences observed in the hydrogen bonding interactions between UDP-Glc and Gal-T1. Based on the present kinetic and crystal structural studies, a possible explanation for the role of LA in the Glc-T activity has been proposed.

Section: The Crystal Structures Of the Complexes Of Gal-t1 And La Witsupporting

confidence: 92%

Section: Discussionsupporting

confidence: 54%

Section: Discussionmentioning

confidence: 75%

See 1 more Smart Citation

α-Lactalbumin (LA) Stimulates Milk β-1,4-Galactosyltransferase I (β4Gal-T1) to Transfer Glucose from UDP-glucose to N-Acetylglucosamine

Ramakrishnan¹,

Shah²,

Qasba³

2001

118

104

“…The conformational changes, from the open to closed state, have been observed in several other glycosyltransferases (38). The important difference between ␤4Gal-T1 and ␤4Gal-T7 in their conformational changes is that the binding of manganese and the UDP molecule alone induces conformational change in ␤4Gal-T7, as observed in the present crystal structure, whereas only manganese and the UDP-Gal molecule, not the UDP molecule, induce conformational change in ␤4Gal-T1 (21,22). The ability of ␤4Gal-T7 to undergo conformational change with the UDP molecule may have been due to the additional hydrogen-bonding interactions between the ␤4Gal-T7 molecule involving Tyr 177 and Arg 250 and the UDP molecule, and they are absent in ␤4Gal-T1 (Fig.…”

Section: Resultssupporting

confidence: 73%

Crystal Structure of the Catalytic Domain of Drosophila β1,4-Galactosyltransferase-7

Ramakrishnan

Qasba

2010

The ␤1,4-galactosyltransferase-7 (␤4Gal-T7) enzyme, one of seven members of the ␤4Gal-T family, transfers in the presence of manganese Gal from UDP-Gal to an acceptor sugar (xylose) that is attached to a side chain hydroxyl group of Ser/Thr residues of proteoglycan proteins. It exhibits the least protein sequence similarity with the other family members, including the well studied family member ␤4Gal-T1, which, in the presence of manganese, transfers Gal from UDP-Gal to GlcNAc. We report here the crystal structure of the catalytic domain of ␤4Gal-T7 from Drosophila in the presence of manganese and UDP at 1.81 Å resolution. In the crystal structure, a new manganese ion-binding motif (HXH) has been observed. Superposition of the crystal structures of ␤4Gal-T7 and ␤4Gal-T1 shows that the catalytic pocket and the substrate-binding sites in these proteins are similar. Compared with GlcNAc, xylose has a hydroxyl group (instead of an N-acetyl group) at C2 and lacks the CH 2 OH group at C5; thus, these protein structures show significant differences in their acceptor-binding site. Modeling of xylose in the acceptor-binding site of the ␤4Gal-T7 crystal structure shows that the aromatic side chain of Tyr 177 interacts strongly with the C5 atom of xylose, causing steric hindrance to any additional group at C5. Because Drosophila Cd7 has a 73% protein sequence similarity to human Cd7, the present crystal structure offers a structure-based explanation for the mutations in human Cd7 that have been linked to Ehlers-Danlos syndrome.

“…A comparison of the K m of UDPGlcNAc, 6 mM, and the K i for UDP, 2 mM, however, reveals a difference in these kinetic parameters, which may reflect subtle differences in the binding of these molecules in the active site. An earlier study used CD spectroscopy to investigate bovine milk galactosyltransferase and determined that only low amounts (10%) of ␣-helix were present (26). A decrease in the amount of ␣-helix was observed when UDP-galactose was added, but no change was observed, however, in the presence of UDP alone in contrast to our data obtained when UDP was added to GlcNAc-T V.…”

Section: Discussioncontrasting

confidence: 66%

Circular Dichroic Spectroscopy of N-Acetylglucosaminyltransferase V and Its Substrate Interactions

Zhang

Peng²,

Chen

et al. 1997

␤-1,6-N-Acetylglucosaminyltransferase V (EC 2.4.1.155) catalyzes the transfer of N-acetylglucosamine (GlcNAc) from UDP-GlcNAc in ␤(1,6)-linkage to the ␣(1,6)-linked mannose of N-linked oligosaccharides. Circular dichroism (CD) was used to investigate the secondary structure of a recombinant, soluble form of the enzyme and its interaction with UDP-GlcNAc and an inhibitory substrate analog. The CD spectrum of the apoenzyme indicated the presence of small amounts of ␤-structure and substantial amounts (>50%) of ␣-helicity. The CD spectra of solutions containing UDP-GlcNAc and different ratios of UDP-GlcNAc:enzyme were measured. Interestingly, the spectrum of each mixture could not be accounted for by simple additivity of the two individual spectra, indicating a change in environment of the chromophores and/or a conformational change of the substrate or protein concomitant with binding. Similar results were obtained with mixtures of UDP and the enzyme. Analysis of the CD difference spectra at three wavelengths yielded an estimated average K d of 4.4 mM for UDP-GlcNAc and 3.8 mM for UDP. By contrast, addition of the CD spectrum of an inhibitory substrate analog of its oligosaccharide acceptor substrate and the CD spectrum of the enzyme could account for that observed of an inhibitor-enzyme mixture; moreover, addition of the inhibitor to a mixture of UDP-GlcNAc and enzyme did not alter the K d associated with UDP-GlcNAc binding to the enzyme. These results and kinetic studies reported herein suggest an ordered reaction in which UDP-GlcNAc binds first to the enzyme, followed by the sequential binding of the trisaccharide substrate.